244
11 Enzymes
as the supernatant after centrifugation at 6000 rpm for 15 min. This crude extract
contains the enzymes mixed with other compounds. Ammonium sulfate at 80%
saturation is then added to precipitate the crude enzyme from the liquid supernatant.
The precipitate is then separated from the rest of the liquid by centrifugation at
10,000 rpm for 15 min. To further purify the enzyme, the solid precipitate is dissolved
in 0.2 M Tris-HCl buffer at pH 8. This solution is then purified by dialysis. This
method can be applied for the extraction of enzymes from waste of fish such as seer
fish sardines, great barracuda, red snapper and milk shark. The protease extracted
using this method has optimum enzyme activity at a pH of 10.
11.5.2 Extraction of Enzyme from Algae
The process of isolating enzymes from algae requires first freeing up the enzymes
from the solid biomass. Like most enzymes, the enzymes present in algae are watersoluble globular proteins. Homogenization of the algae biomass followed by separation of the liquid and solid by centrifugation will yield a liquid supernatant
which comprises a mixture of some soluble polysaccharides and other proteins. The
enzymes can then be precipitated out of the solution. This is then redissolved and
further purified using methods like gel filtration (Mogharabi and Faramarzi 2016).
In one example extraction process reported by Bele et al. (2014a, b), the algae
biomass is collected and washed with water to remove debris. It is then dried and
ground into powdered form. The powdered algae biomass is mixed with distilled
water to allow all water-soluble components to dissolve. The liquid is then separated
from the solid mass. The protein is precipitated out of the aqueous phase using
ammonium sulfate added at a mass-to-volume ratio of 0.0663 g:1 ml. The solid
protein precipitate is then recovered by centrifugation at 10,000 g for 15 min at 4 °C.
The solid fraction contains the enzyme. This is then redissolved in trichloroacetic acid
(TCA) buffer and further purified by gel filtration using Sephadex grade 100 (Bele
et al. 2014a, b). This method was used to extract enzymes from Ulva lactuca, Ulva
fasciata, Chaetomorpha antenna, Gelidium pusillum and Enteromorpha compressa.
Magnesium sulfate has been conventionally used for precipitation of globular
proteins for several decades (Howe 1921). Other salts such as sodium sulfate and
magnesium sulfate can also be used for similar purposes. The choice of salt depends
on the requirements of the processor. For example, protein precipitates formed using
magnesium sulfate are gelatinous in nature and this results in relatively slow filtration; sodium sulfate is preferred when working at temperatures above 34 °C, and
ammonium sulfate can be used where the nitrogen element in the salt does not pose
a challenge in processing or analysis.
Enzyme extraction from marine algae can also be carried out using two-phase
extraction with PEG-4000 (Bele et al. 2014a). In this method, sodium sulfate is
added to the supernatant of the centrifuged sample 0.75 g in 10 ml mass of sample per
volume of salt solution. To this, ~3 ml of PEG-4000 is added at a 50% concentration.
11 Enzymes
as the supernatant after centrifugation at 6000 rpm for 15 min. This crude extract
contains the enzymes mixed with other compounds. Ammonium sulfate at 80%
saturation is then added to precipitate the crude enzyme from the liquid supernatant.
The precipitate is then separated from the rest of the liquid by centrifugation at
10,000 rpm for 15 min. To further purify the enzyme, the solid precipitate is dissolved
in 0.2 M Tris-HCl buffer at pH 8. This solution is then purified by dialysis. This
method can be applied for the extraction of enzymes from waste of fish such as seer
fish sardines, great barracuda, red snapper and milk shark. The protease extracted
using this method has optimum enzyme activity at a pH of 10.
11.5.2 Extraction of Enzyme from Algae
The process of isolating enzymes from algae requires first freeing up the enzymes
from the solid biomass. Like most enzymes, the enzymes present in algae are watersoluble globular proteins. Homogenization of the algae biomass followed by separation of the liquid and solid by centrifugation will yield a liquid supernatant
which comprises a mixture of some soluble polysaccharides and other proteins. The
enzymes can then be precipitated out of the solution. This is then redissolved and
further purified using methods like gel filtration (Mogharabi and Faramarzi 2016).
In one example extraction process reported by Bele et al. (2014a, b), the algae
biomass is collected and washed with water to remove debris. It is then dried and
ground into powdered form. The powdered algae biomass is mixed with distilled
water to allow all water-soluble components to dissolve. The liquid is then separated
from the solid mass. The protein is precipitated out of the aqueous phase using
ammonium sulfate added at a mass-to-volume ratio of 0.0663 g:1 ml. The solid
protein precipitate is then recovered by centrifugation at 10,000 g for 15 min at 4 °C.
The solid fraction contains the enzyme. This is then redissolved in trichloroacetic acid
(TCA) buffer and further purified by gel filtration using Sephadex grade 100 (Bele
et al. 2014a, b). This method was used to extract enzymes from Ulva lactuca, Ulva
fasciata, Chaetomorpha antenna, Gelidium pusillum and Enteromorpha compressa.
Magnesium sulfate has been conventionally used for precipitation of globular
proteins for several decades (Howe 1921). Other salts such as sodium sulfate and
magnesium sulfate can also be used for similar purposes. The choice of salt depends
on the requirements of the processor. For example, protein precipitates formed using
magnesium sulfate are gelatinous in nature and this results in relatively slow filtration; sodium sulfate is preferred when working at temperatures above 34 °C, and
ammonium sulfate can be used where the nitrogen element in the salt does not pose
a challenge in processing or analysis.
Enzyme extraction from marine algae can also be carried out using two-phase
extraction with PEG-4000 (Bele et al. 2014a). In this method, sodium sulfate is
added to the supernatant of the centrifuged sample 0.75 g in 10 ml mass of sample per
volume of salt solution. To this, ~3 ml of PEG-4000 is added at a 50% concentration.